Bottom Line:
While Clb1 phosphorylation is dependent on activity of both CDK and polo-like kinase Cdc5, its nuclear localization requires CDK but not Cdc5 activity.Furthermore we show that increased nuclear localization of Clb1 during meiosis enhances activation of FEAR (Cdc Fourteen Early Anaphase Release) pathway.We discuss the significance of our results in relation to regulation of exit from meiosis I.

ABSTRACTCyclin-dependent kinases (CDK) are master regulators of the cell cycle in eukaryotes. CDK activity is regulated by the presence, post-translational modification and spatial localization of its regulatory subunit cyclin. In budding yeast, the B-cyclin Clb1 is phosphorylated and localizes to the nucleus during meiosis I. However the functional significance of Clb1's phosphorylation and nuclear localization and their mutual dependency is unknown. In this paper, we demonstrate that meiosis-specific phosphorylation of Clb1 requires its import to the nucleus but not vice versa. While Clb1 phosphorylation is dependent on activity of both CDK and polo-like kinase Cdc5, its nuclear localization requires CDK but not Cdc5 activity. Furthermore we show that increased nuclear localization of Clb1 during meiosis enhances activation of FEAR (Cdc Fourteen Early Anaphase Release) pathway. We discuss the significance of our results in relation to regulation of exit from meiosis I.

pone-0079001-g002: Clb1 modification and nuclear localisation are meiosisâ€“specific.PMET3CDC20 CLB1-myc9 cells were arrested in metaphase by transferring them to YEPD medium with 0.15% methionine for 2.5 hours. Culture samples were taken for in situ immunofluorescence and for preparing whole cell extracts. Localisation of Clb1-Myc and spindle formation was examined by in situ immunofluorescence. A) Percentage of cells containing metaphase spindles following addition of methionine is indicated. B) Representative image of a cell arrested in metaphase displaying Clb1 localisation is shown. C) Localization of Clb1 during the experiment is graphically presented (green-nuclear, red-cytoplasmic, blue-no signal). D) Whole cell extracts were subjected to SDS-PAGE followed by Western analysis for assaying Clb1 phosphorylation. For comparison, whole cell extract from diploid PCLB2CDC20 CLB1-myc9 cells after 8 hours in SPM was included in the gel.

Mentions:
We then tested whether Clb1 modification and nuclear localisation also occur during a mitotic metaphase arrest. PMET3CDC20 cells expressing a TAP tagged version of Clb1 were arrested in metaphase by Cdc20 depletion, 80% of cells had metaphase spindles 2.5 hours after resuspension in medium containing methionine, indicating that a majority of cells were arrested in metaphase (Figure 2A). However Clb1 was neither modified nor enriched in the nucleus (Figure 2Bâ€“D). Thus, phosphorylation and nuclear localization of Clb1 are meiosis-specific.

pone-0079001-g002: Clb1 modification and nuclear localisation are meiosisâ€“specific.PMET3CDC20 CLB1-myc9 cells were arrested in metaphase by transferring them to YEPD medium with 0.15% methionine for 2.5 hours. Culture samples were taken for in situ immunofluorescence and for preparing whole cell extracts. Localisation of Clb1-Myc and spindle formation was examined by in situ immunofluorescence. A) Percentage of cells containing metaphase spindles following addition of methionine is indicated. B) Representative image of a cell arrested in metaphase displaying Clb1 localisation is shown. C) Localization of Clb1 during the experiment is graphically presented (green-nuclear, red-cytoplasmic, blue-no signal). D) Whole cell extracts were subjected to SDS-PAGE followed by Western analysis for assaying Clb1 phosphorylation. For comparison, whole cell extract from diploid PCLB2CDC20 CLB1-myc9 cells after 8 hours in SPM was included in the gel.

Mentions:
We then tested whether Clb1 modification and nuclear localisation also occur during a mitotic metaphase arrest. PMET3CDC20 cells expressing a TAP tagged version of Clb1 were arrested in metaphase by Cdc20 depletion, 80% of cells had metaphase spindles 2.5 hours after resuspension in medium containing methionine, indicating that a majority of cells were arrested in metaphase (Figure 2A). However Clb1 was neither modified nor enriched in the nucleus (Figure 2Bâ€“D). Thus, phosphorylation and nuclear localization of Clb1 are meiosis-specific.

Bottom Line:
While Clb1 phosphorylation is dependent on activity of both CDK and polo-like kinase Cdc5, its nuclear localization requires CDK but not Cdc5 activity.Furthermore we show that increased nuclear localization of Clb1 during meiosis enhances activation of FEAR (Cdc Fourteen Early Anaphase Release) pathway.We discuss the significance of our results in relation to regulation of exit from meiosis I.

ABSTRACTCyclin-dependent kinases (CDK) are master regulators of the cell cycle in eukaryotes. CDK activity is regulated by the presence, post-translational modification and spatial localization of its regulatory subunit cyclin. In budding yeast, the B-cyclin Clb1 is phosphorylated and localizes to the nucleus during meiosis I. However the functional significance of Clb1's phosphorylation and nuclear localization and their mutual dependency is unknown. In this paper, we demonstrate that meiosis-specific phosphorylation of Clb1 requires its import to the nucleus but not vice versa. While Clb1 phosphorylation is dependent on activity of both CDK and polo-like kinase Cdc5, its nuclear localization requires CDK but not Cdc5 activity. Furthermore we show that increased nuclear localization of Clb1 during meiosis enhances activation of FEAR (Cdc Fourteen Early Anaphase Release) pathway. We discuss the significance of our results in relation to regulation of exit from meiosis I.